Proteomics Technology Improvement And The Study Of HCC

Objectives Proteomics research including sample extraction and purification of early; protein separation and identification of interim analysis; data processing and analysis of late. Protein identification compared with gene sequencing, the main difficulty is the structure and composition of proteins than genes complex and proteome is dynamic, so one of the limiting factors identified by mass spectrometry technology for high-throughput protein is a protein separation technology Traditional proteomics research strategy is based on two-dimensional electrophoresis-mass spectrometry (2-DE-MS), finite-dimensional electrophoretic separation capabilities, and liquid chromatography (LC) to make up for the two-dimensional gel electrophoresis of defects, thus we need explore new for complex biological samples protein digests and separation technology, to improve the level of proteins identified by mass spectrometry. Neoplastic diseases has been considered from the perspective of proteomics is a protein deficiency, which occurs at different stages of development, even in the absence of any symptoms of early precancerous lesions, the levels of protein in the serum has often occurred changes. The new diagnostic technologies and the integration of proteomics methods, used in clinical oncology diseases, verify the feasibility of a certain body fluids in complex proteomics research.Methods1,With promega and Self-owned trypsin digestion of different concentrations of bull serum albumin(BSA) measured total ion chromatogram (TIC) and using Proteome Discoverer software for data processing, measurement BSA coverage rate.2,Digested with Self-owned trypsin BSA, two-dimensional liquid enzyme used in this study will be prepared in one of the capillary column were separated by high performance liquid chromatography and mass spectrometry (nanoHPLC-MS) technology platform, through TIC, retention time (RT) and signal strength, examine its capillary column separation performance and reproducibility.3, Select from March2012to June2013in Tianjin Third Central Hospital for medical examination of30cases of healthy people, and hepatobiliary surgery for treatment of20cases of HBV-related hepatitis patients,20cases of HBV-related cirrhosis patients and20serum liver cancer before surgery in patients with liver cells, which is included in the standard serum of patients hospitalized at the beginning of treatment has not been any morning fasting serum samples, with nanoHPLC-MS techniques for protein identification and analysis of pre-treatment, the resulting data will be processed using software OMSSA for analysis, the number of proteins identified for follow-up screening of potential protein markers to prepare.Results1, With promega and Self-owned trypsin digestion of different concentrations of BSA, under the same experimental conditions, nanoHPLC-MS technique identified Self-owned trypsin digestion of high, medium and low concentrations of BSA coverage of38.22%,28.34%,13.18%; while using promega tryptic BSA coverage of34.93%,26.36%,10.21%.2, The Self-owned trypsin and one-dimensional capillary column for the identification of BSA and analyzed by LTQ-Orbitrap XL mass spectrometer, which at very low concentrations, identification BSA coverage of24.22%.3,Under the same experimental conditions, the observed one-dimensional capillary column reproducibility. Digested BSA peptides selected mixture of mass-to-charge ratio (m/z) of740.4peptides, were twice RT51.90min,51.95min signal strength are respectively6.77×105,7.42×105.4, By proteomics technology to improve and integrate the use of Self-owned trypsin digested proteins, with nanoHPLC-MS technology platform, using one-dimensional capillary column for separation and LTQ Orbitrap XL mass spectrometer accurate quantification of proteins, and through OMSSA software protein analysis of the data, the final identification of3,578kinds of proteins.Conclusions Through statistical analysis of physical and chemical properties BSA, experimental results show a high catalytic activity of Self-owned trypsin, one-dimensional capillary column has a better separation performance and reproducibility. This study will be applied to improve the technology and integrated mode in human serum samples, after Self-owned trypsin digestion, one-dimensional capillary column separation, LTQ Orbitrap XL accurate quantification of protein mass, and then through the OMSSA software analysis data, the final identification of3,578kinds of proteins. Identification of the protein sufficient to support the number of post-data processing, screening of potential marker protein expression differences. Looking for protein markers for subsequent development of liver tumors have occurred in the early diagnosis, monitoring the value of a sound foundation. This method can be inferred with some feasibility studies in proteomics, can be liquid expression profiling to build mass spectrometry system for complex fluids, cells or tissues, such as the complexity of the proteome.